Modulation of excitable tissue function by peripherally administered erythropoietin

ABSTRACT

Methods and compositions are provided for protecting or enhancing excitable tissue function in mammals by systemic administration of an erythropoietin receptor activity modulator, such as erythropoietin, which signals via an EPO-activated receptor to modulate the function of excitable tissue. Excitable tissues include central neuronal tissues, such as the brain, peripheral neuronal tissues, retina, and heart tissue. Protection of excitable tissues provides treatment of hypoxia, seizure disorders, neurodegenerative diseases, hypoglycemia, and neurotoxin poisoning. Enhancement of function is useful in learning and memory. The invention is also directed to compositions and methods for facilitating the transport of molecules across endothelial cell tight junction barriers, such as the blood-brain barrier, by association of molecules with an erythropoietin receptor activity modulator, such as an erythropoietin.

This application is a continuation application under 35 U.S.C. § 120 ofU.S. application Ser. No. 09/717,057, filed Nov. 21, 2000, which is adivisional of U.S. application Ser. No. 09/547,220, filed Apr. 11, 2000,now abandoned, which claims benefit under 35 U.S.C. § 119(e) of U.S.provisional patent application No. 60/129,131, filed Apr. 13, 1999, theentire contents of each of which is incorporated herein by reference inits entirety.

1. FIELD OF THE INVENTION

The present invention is directed to the use of peripherallyadministered erythropoietin and other erythropoietin receptor activitymodulators or EPO-activated receptor modulators to positively affectexcitable tissue function. This includes the protection of excitabletissue, such as neuronal and cardiac tissue, from neurotoxins, hypoxia,and other adverse stimuli, and the enhancement of excitable tissuefunction, such as for facilitating learning and memory. The presentinvention is further drawn to methods for transport of substances acrossendothelial cell barriers by association with an erythropoietinmolecule, erythropoietin receptor activity modulator or otherEPO-activated receptor modulators.

2. BACKGROUND OF THE INVENTION

Various acute and chronic conditions and diseases originate fromexcitable tissue damage and dysfunction brought about by external andinternal stimuli. Such stimuli include lack of adequate oxygenation orglucose, neurotoxins, consequences of aging, infectious agents, andtrauma. For example, excitable tissue may be subjected to damage as aconsequence of seizures and chronic seizure disorders, convulsions,epilepsy, stroke, Alzheimer's disease, Parkinson's disease, centralnervous system injury, hypoxia, cerebral palsy, brain or spinal cordtrauma, AIDS dementia and other forms of dementia, age-related loss ofcognitive function, memory loss, amyotrophic lateral sclerosis, multiplesclerosis, hypotension, cardiac arrest, neuronal loss, smoke inhalationand carbon monoxide poisoning.

It is widely understood that decreases in energy supply available to thebrain, such as glucose or oxygen, results in a profound impairment ofbrain function, including cognition. Many (but not all) neurons in thecentral nervous system are easily damaged while working undermetabolically-limited conditions, e.g., hypoxia, hypoglycemia, stress,and/or prolonged, strong excitation. Under these circumstances, theelectrochemical gradients of these cells often collapse, resulting inirreversible neuronal injury and cell death. Current opinion favors thisgeneral mechanism as a common final pathway for a wide range of commonand debilitating degenerative neurological diseases including stroke,epilepsy, and Alzheimer's disease.

Although the consequences of limited energy substrate on brain functionare well known, the effects of improving energy delivery in an otherwisenormal brain has been less extensively explored. Current data suggeststrongly that improved delivery of either glucose or oxygen markedlyimproves complex cognitive function in both animal models and in normalhuman subjects (Kopf et al., 1994, Behavioral and Neural Biology62:237-243; Li et al., 1998, Neuroscience 85:785-794; Moss et al., 1996,Psychopharmacology 124:255-260). Further, a growing list ofneuropeptides produced within the brain have been demonstrated todirectly provide an improvement in cognitive function in normal brain.The physiological basis of these enhancements ultimately depends uponremodeling of neuronal interconnections through synaptic changes.

Brain tissue cytoarchitecture exhibits extreme plasticity and undergoescontinuous remodeling. These processes, mediated by many trophicmolecules, occur not only following injury, but also play a prominentrole in learning, memory, and cognitive function. Although the prototypeneurotrophin is nerve growth factor (NGF), an increasing number ofcytokines have been recognized to perform trophic functions in the brain(Hefti et al. 1997, Annu. Rev. Pharmacol. Toxicol. 37:239-67).

Recently, a number of independent investigators have recognized thatnervous tissue expresses high levels of both EPO and its receptor(EPO-R; Digicaylioglu et al., 1998, Proc. Natl. Acad. Sci. USA92:3717-20; Juul et al., Pediatr. Res. 43:40-9; Marti et al., 1997,Kidney Int. 51:416-8; Morishita et al., 1997, Neuroscience 76:105-16).Although it appears that EPO and its receptor proteins are each theproducts of single genes, the CNS versions are significantly smaller.The physiological meaning of this observation has not been clarified,but the mass differences do appear to modify biological activity. Forexample, in studies of human patients, investigators have concluded thatEPO is not transported into the brain from the periphery (Marti et al.,1997, supra). To date, however, this possibility has not been evaluatedfor EPO by any direct study. Although brain EPO is about 15% smallerthan renal EPO (due to differences in sialylation), brain EPO is moreactive in erythroid colony stimulation at low ligand concentrations(Masuda et al., 1994, J. Biol. Chem. 269:19488-93). On the other hand,the CNS receptor exhibits a much lower affinity for deglycosylated EPOthan the 30% larger peripheral receptor (Konishi et al., 1993, BrainRes. 609:29-35; (Masuda et al., 1993, J. Biol. Chem. 268:11208-16).

In the brain, EPO expression has been found in astrocytes, and increasedEPO expression and release can be induced by hypoxia and other metabolicstressors (Marti et al., 1996, Eur. J. Neurosci. 8:666-76; Masuda etal., 1993, J. Biol. Chem. 268:11208-16; Masuda et al., 1994, J. Biol.Chem. 269:19488-93) or even by occupancy of other receptors such asinsulin-like growth factor family (Masuda et al., 1997, Brain Res.746:63-70). Neurons are one target for this secreted EPO as they expressEPO-R in a highly cell type-specific manner (Morishita et al., 1997,Neuroscience 76:105-16). In contrast to EPO itself, EPO-R density doesnot appear to be modulated during metabolic stress (Digicaylioglu etal., 1995, Proc. Natl. Acad. Sci. USA 92:3717-20).

Recent study has demonstrated that EPO impressively protects againsthypoxic neuronal injury in vitro, as well as in vivo when injecteddirectly into the cerebral ventricles (Morishita et al., 1997,Neuroscience 76:105-16; Sadamoto et al., 1998, Biochem. Biophys. Res.Commun. 253:26-32; Sakanaka et al., 1998, Proc. Natl. Acad. Sci. USA95:4635-40). Konishi et al. (1993, Brain Res. 609:29-35) havedemonstrated that EPO promotes the in vivo survival of cholinerpicneurons in adult rats when injected directly into the cerebralventricles. EPO administered centrally into the cerebral ventricles alsosuccessfully prevents ischemic injury-related deficits in spatiallearning in rats (Sadamoto et al., 1998, Biochem. Biophys. Res. Commun.253:26-32). A recent publication suggests that only a 17-amino acidportion of EPO is needed for these neurotrophic effects in culturedneural cells (Campana et al., 1998, Int. J. Mol. Med. 1:235-41).

For many years, the only clear physiological role of erythropoietin(EPO) had been its control of the production of red blood cells.Recently, several lines of evidence suggest that EPO, as a member of thecytokine superfamily, performs other important physiologic functionswhich are mediated through interaction with the erythropoietin receptor(EPO-R). These actions include mitogenesis, modulation of calcium influxinto smooth muscle and neural cells, and effects on intermediarymetabolism. It is believed that EPO provides compensatory responses thatserve to improve hypoxic cellular microenvironments. Although studieshave established that EPO injected intracranially protects neuronsagainst hypoxic neuronal injury, intracranial administration is animpractical and unacceptable route of administration for therapeuticuse, particularly for normal individuals. Furthermore, previous studiesof anemic patients given EPO have concluded thatperipherally-administered EPO is not transported into the brain (Martiet al., 1997, supra).

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

3. BRIEF SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods formodulating excitable tissue function in mammals, as well as methods andcompositions for drug delivery to excitable tissues. The invention isbased, in part, on the Applicants' discovery that erythropoietin (EPO),administered systemically and at a high dosage, is specifically taken upby the brain. In particular, the Applicants have found that EPO,delivered in high doses, can cross the blood-brain barrier, where it canenhance cognitive function, and protect neural tissue from damageresulting from stressful conditions, such as hypoxia Erythropoietin andEPO, used interchangeably herein, and EPO receptor activity modulators,and EPO-activated receptor modulators refer to compounds, which, whenadministered systemically (outside the blood-brain barrier), are capableof activating EPO-activated receptors of electrically excitable tissuesto enhance and/or protect from injury and death. Thus, EPO can refer toany form of erythropoietin that can modulate excitable tissue, as wellas EPO analogs, fragments and mimetics thereof. In a preferredembodiment, for use in the methods of the present invention, theerythropoietin displays increased specificity for the brain EPOreceptor. In another embodiment, the erythropoietin isnonerythropoietic. In yet another embodiment, the erythropoietin isadministered at a dose greater than the dose necessary to maximallystimulate erythropoiesis.

The present invention provides a pharmaceutical composition in dosageunit form adapted for modulation of excitable tissue, enhancement ofcognitive function or delivery of compounds across endothelial tightjunctions which comprises, per dosage unit, an effective non-toxicamount within the range from about 50,000 to 500,000 Units of EPO, anEPO receptor activity modulator, an EPO-activated receptor modulator, ora combination thereof, and a pharmaceutically acceptable carrier. In oneembodiment, the effective non-toxic amount of EPO in said pharmaceuticalcomposition comprises 50,000 to 500,000 Units of EPO. In anotherembodiment, the effective non-toxic amount of EPO of said pharmaceuticalpreparation is a dose effective to achieve a circulating level of EPO ofgreater than 10,000 mU/ml of serum. In another embodiment, thecirculating level of EPO is achieved about 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 hours after the administration of EPO. In another embodiment, theinvention provides a pharmaceutical kit comprising an effective amountof EPO for modulation of excitable tissue, enhancement of cognitivefunction or delivery of compounds across endothelial tight junctionspackaged in one or more containers.

The present invention provides a method for modulating the function ofexcitable tissue in a mammal, comprising administering peripherally tosaid mammal an effective amount of an erythropoietin. The excitabletissue may be normal tissue or abnormal, diseased tissue. In oneembodiment, the excitable tissue is neuronal tissue of the centralnervous system. In other embodiments, the excitable tissue is selectedfrom the group consisting of neuronal tissue of the peripheral nervoussystem and heart tissue.

In one embodiment, a method is provided for the enhancement of excitabletissue function in a mammal, in particular, both normal and abnormal,excitable tissue, by administering peripherally an effective amount ofEPO or an EPO receptor activity modulator. Enhancement of excitabletissue function provides enhancement of, for example, learning,associative learning, or memory. Non-limiting examples of conditions ordiseases treatable by this aspect of the present invention include mooddisorders, anxiety disorders, depression, autism, attention deficithyperactivity disorder, Alzheimer's disease, aging and cognitivedysfunction.

In another embodiment, the modulation of excitable tissue providesprotection from pathology resulting from injury to excitable tissue, forexample, to neurons of the central nervous system, peripheral nervoussystem, or heart tissue. Such pathology may result from injuriesincluding, but not limited to hypoxia, seizure disorders,neurodegenerative diseases, neurotoxin poisoning, multiple sclerosis,hypotension, cardiac arrest, radiation, or hypoglycemia. In oneembodiment, the pathology is a result of hypoxia, and may be prenatal orpostnatal oxygen deprivation, suffocation, choking, near drowning,post-surgical cognitive dysfunction, carbon monoxide poisoning, smokeinhalation, chronic obstructive pulmonary disease, emphysema, adultrespiratory distress syndrome, hypotensive shock, septic shock, insulinshock, anaphylactic shock, sickle cell crisis, cardiac arrest,dysrhythmia or nitrogen narcosis. In the instance wherein the pathologyis a seizure disorder, it may be, by way of non-limiting example,epilepsy, convulsions or chronic seizure disorder. In the instancewherein the pathology is a neurodegenerative disease, it may be, forexample, stroke, Alzheimer's disease, Parkinson's disease, cerebralpalsy, brain or spinal cord trauma, AIDS dementia, age-related loss ofcognitive function, memory loss, amyotrophic lateral sclerosis, seizuredisorders, alcoholism, retinal ischemia, aging, glaucoma or neuronalloss. In another embodiment, administration of EPO may be used toprevent injury or tissue damage during surgical procedures, such as, forexample, tumor resection or aneurysm repair.

In yet another embodiment, methods are provided for facilitating thetranscytosis of a molecule across an endothelial cell barrier in amammal by administration of a composition of a molecule in associationwith erythropoietin. The association between the molecule to betransported and EPO may be, for example, a labile covalent bond, astable covalent bond, or a noncovalent association with a binding sitefor the molecule. In one embodiment, the endothelial cell barriers maybe the blood-brain barrier, the blood-eye barrier, the blood-testesbarrier, the blood-ovary barrier or the blood-placenta barrier.

The invention further provides a composition for transporting a moleculevia transcytosis across an endothelial cell barrier comprising saidmolecule in association with an EPO, an EPO receptor activity modulator,or an EPO-activated receptor modulator. In one embodiment, the EPO iserythropoietin, an erythropoietin analog, an erythropoietin mimetic, anerythropoietin fragment, a hybrid erythropoietin molecule, anerythropoietin receptor-binding molecule, an erythropoietin agonist, arenal erythropoietin, a brain erythropoietin, an oligomer thereof, amultimer thereof, a mutein thereof, a congener thereof, anaturally-occurring form thereof, a synthetic form thereof, arecombinant form thereof, or a combination thereof. In anotherembodiment, the molecule of said composition is a hormone, aneurotrophic factor, an antimicrobial agent, a radiopharmaceutical, anantisense compound, an antibody, an immunosuppressant, a toxin, or ananti-cancer agent.

Suitable molecules for transport by the method of the present inventioninclude, but are not limited to hormones, such as growth hormone,antibiotics, anti-cancer agents, and toxins.

These and other aspects of the present invention will be betterappreciated by reference to the following Figures and DetailedDescription.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B. Morris Water Maze test A. The results of a Morris Water Mazetest performed in mice receiving either EPO or saline (SHAM)administered peripherally each day. B. Subjects receiving EPO performedsignificantly better than SHAM treated subjects. The regression line(R²=0.88) shows a slope (0.68) significantly different from a slope of1, markedly in favor of the EPO group.

FIGS. 2A-C. Conditioned Taste Aversion test A. Comparison of peripheralsham and EPO treatment on water consumption in mice undergoingConditioned Taste Aversion testing. Water consumption is expressed as apercentage of the volume consumed by control mice, which were not madeill with lithium chloride. B and C illustrate that the EPO-enhancedlearning is robust, as EPO subjects tolerated much greater thirst thancontrols in avoidance of water containing the illness-associated cue yetspent more time seeking water.

FIG. 3A-B A. The results of an experiment which demonstrates thatperipherally-administered EPO pretreatment reduces seizure severity andprotects mice from convulsions and death by the neurotoxin kainate. Thenumbers in parentheses under each column indicate the number of animalsreceiving each kainate dose. B shows that the protective effects ofperipherally-administered EPO increase with daily administration of EPO.C illustrates that the onset of action of EPO is delayed, characteristicof the induction of a gene expression program.

FIG. 4A-B depicts the protective effect of rhEPO against ischemic braininjury (focal stroke). A. Systemic administration of EPO given atvarious times after the induction of brain ischemia reduces infarctsize. B. Comparison of two forms of EPO in protecting brain from injuryin this model: recombinant human (rhEPO) and 17 amino acid EPOderivative (17-mer) illustrates that some EPO analogs are ineffectivefor neuroprotection.

FIG. 5 depicts the protective effect of rhEPO against blunt traumadelivered to the cerebral cortex.

FIG. 6A-B depicts the protective effect of EPO from ischemic heartinjury. A. Creatine kinase (CK) activity, an indicator of damage to themyocardial cells. B. Myeloperoxidase (MPO) activity, a measure ofinflammation.

FIG. 7 shows that treatment of mice with EPO delays and reduces theneurological symptoms produced by an experimental allergic encephalitis,a model of multiple sclerosis.

FIG. 8A-B A. The minimum effective dose of EPO to provideneuroprotection in a focal stroke model performed in rats. B. Serumlevels of EPO at various time points after 5000 U of rhEPO wasadministered intraperitoneally to female Balb/c mice.

FIG. 9A-C A. Immunolocalization of EPO-R on and around capillaries. B.Biotinylated EPO administered IP to mice is found at 5 hours within thebrain immediately surrounding capillaries. C. After 17 hours, the biotinlabel can be found to be within specific neurons.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for the use oferythropoietin (EPO) for modulating excitable tissue function, such as,for example, enhancing cognitive function and protecting excitable cellsfrom toxic stimuli. In particular, the invention provides compositionscomprising EPO, as well as methods for their use in prophylactic andtherapeutic treatments, including drug delivery. As used herein,excitable tissue, includes, but is not limited to, neuronal tissue ofthe central and peripheral nervous systems, and cardiac tissue.

The invention described herein provides methods for modulating excitabletissue function by peripheral administration of EPO, or an EPO receptoractivating molecule or a molecule exhibiting EPO-activated receptoractivity, as well as any molecule that mimics the activity of EPO byacting through other, non-classical EPO receptors. Without being boundby any particular mechanism of action, such a molecule may signal viathe EPO receptor, for example, initiates a signal transduction cascadeultimately activating a gene expression program resulting in theprotection or enhancement of excitable tissue function. Moleculescapable of interacting with the EPO receptor and modulating the activityof the receptor, herein referred to as EPO or EPO receptor activitymodulators, are useful in the context of the present invention for theprotection or enhancement of excitable tissue function. These moleculesmay be, for example, naturally-occurring, synthetic, or recombinantforms of EPO molecules, describe above, or other molecules which may notnecessarily resemble EPO in any manner, except to modulate EPO receptoractivity, as described herein. These molecules may be used incombination for the various purposes herein described.

The compositions and methods described herein can be used to treatand/or protect both normal tissue or abnormal tissue, for example,neurons of the central nervous system, neurons of the peripheral nervoussystem, or heart tissue. In particular, in Section 5.1, below, EPOcompositions useful for practice with invention are described. InSection 5.2.1, methods are described for the use of such EPOcompositions for enhancing the function of excitable tissue, such aslearning, memory, and other aspects of cognitive function, and, inSection 5.2.2, methods for protecting excitable tissue from damage andinjury are described. Also described in Section 5.2.3 below, thediscovery of the unexpected ability of EPO to cross capillaryendothelial cell tight junctions provides methods for delivery ofcompounds across such barriers. Finally, described in Section 5.3 areconditions that can be targeted using the methods of the invention, andin Section 5.4, methods of administration and effective dosages of suchEPO compositions are described.

5.1 Compositions Comprising Erythropoietin

EPO compositions suitable for use with the invention include anyerythropoietin compound that, when administered peripherally, is capableof activating EPO-activated receptors to modulate, i.e. enhance thefunction of, protect from damage or injury, or deliver compounds to,excitable tissue. Erythropoietin is a glycoprotein hormone which inhumans has a molecular weight of 34 to 38 kD. The mature proteincomprises 166 amino acids, and the glycosyl residues comprise about 40%of the weight of the molecule. The forms of EPO useful in the practiceof the present invention encompass naturally-occurring, synthetic andrecombinant forms of the following molecules: erythropoietin,erythropoietin analogs, erythropoietin mimetics, erythropoietinfragments, hybrid erythropoietin molecules, erythropoietinreceptor-binding molecules, erythropoietin agonists, renalerythropoietin, brain erythropoietin, oligomers and multimers thereof,muteins thereof, and congeners thereof. The term “erythropoietin” and“EPO” may be used interchangeably or conjunctively.

Synthetic and recombinant molecules, such as brain EPO and renal EPO,recombinant mammalian forms of EPO, as well as its naturally-occurring,tumor-derived, and recombinant isoforms, such as recombinantly-expressedmolecules and those prepared by homologous recombination are providedherein. Furthermore, the present invention includes molecules includingpeptides which bind the EPO receptor, as well as recombinant constructsor other molecules which possess part or all of the structural and/orbiological properties of EPO, including fragments and multimers of EPOor its fragments. EPO herein embraces molecules with altered EPOreceptor binding activities, preferably with increased receptoraffinity, in particular as pertains to enhancing transport acrossendothelial cell barriers. Muteins comprising molecules which haveadditional or reduced numbers of glycosylation sites are includedherein. As noted above, the terms “erythropoietin,” “EPO,” and“mimetics” as well as the other terms are used interchangeably herein torefer to the excitable tissue protective and enhancing molecules relatedto EPO as well as the molecules which are capable of crossingendothelial tight junctions and as such are useful as a delivery meansfor other molecules. Furthermore, molecules produced by transgenicanimals are also encompassed here. It should be noted that EPO moleculesas embraced herein do not necessarily resemble EPO structurally or inany other manner, except for ability to interact with the EPO receptoror modulate EPO receptor activity or activate EPO-activated signalingcascades, as described herein.

By way of non-limiting example, forms of EPO useful for the practice ofthe present invention include EPO muteins, such as those with alteredamino acids at the carboxy terminus described in U.S. Pat. No. 5,457,089and in U.S. Pat. No. 4,835,260; EPO isoforms with various numbers ofsialic acid residues per molecule, such as described in U.S. Pat. No.5,856,292; polypeptides described in U.S. Pat. No. 4,703,008; agonistsdescribed in U.S. Pat. No. 5,767,078; peptides which bind to the EPOreceptor as described in U.S. Pat. Nos. 5,773,569 and 5,830,851;small-molecule mimetics which activate the EPO receptor, as described inU.S. Pat. No. 5,835,382; and EPO analogs described in WO 9505465, WO9718318, and WO 9818926. All of the aforementioned citations areincorporated herein to the extent that such disclosures refer to thevarious alternate forms or processes for preparing such forms of theerythropoietins of the present invention. EPO can be obtainedcommercially (under the trademarks of PROCRIT, available from OrthoBiotech, and EPOGEN, available from Amgen, Inc., Thousand Oaks, Calif.).

In a further embodiment of the present invention, the EPO moleculesembraced herein include hybrid EPO molecules that may be prepared whichcomprise the EPO receptor modulating activity as well as anotheractivity, for example, that of growth hormone. Such hybrid moleculeswith multiple domains thus possess the ability to interact with the EPOreceptor—as well as having the activity of another molecule such as ahormone. Methods of preparation of such molecules with two domains areknown to one skilled in the art. As will be described in more detail inSection 5.2.3 below, one feature of such molecules is transport acrossendothelial cell barriers provided by the EPO receptor activitymodulating domain, and activity of the other molecule at the targetsite.

Any of the compounds described above may be tested to identify EPOcompounds capable of modulating excitable tissue, i.e. enhance thefunction of, protect from damage or injury, or deliver compoundsthereto, using the assays described herein. For example, EPO compoundsmay be tested for their ability to enhance the function of excitabletissue, such as learning, memory, and other aspects of cognitivefunction using the methods described in Section 5.2.1. Examples of invivo assays for cognitive function include the Morris Water Maze test,an example of which is described in Section 6, and the Conditioned TasteAversion test, an example of which is described in detail in Section 7.In addition, the EPO compounds described above may be tested usingassays described in Section 5.2.2, to identify EPO compounds capable ofprotecting excitable tissue from damage and injury. The Examplesdescribed in Sections 8, 9, 10, 11, and 12 provide specific examples ofsuch assays. EPO compounds may also be assayed for their capacity todelivery of compounds across epithelial tight junctions, such as theblood-brain barrier, using assays such as those described in Section5.2.3 and Section 9, below. Thus, EPO compositions suitable for use withthe invention include any and all compounds that, when administeredperipherally, are capable of signaling through EPO-activated receptorsto modulate excitable tissue, i.e. enhance the function of, protect fromdamage or injury, or deliver compounds thereto.

5.2 Methods for Prophylactic and Therapeutic Use of the Invention

In various embodiments of the invention, EPO compositions can be usedfor protecting excitable tissue from injury or hypoxic stress, enhancingthe function of excitable tissue, or for delivery of compounds acrossendothelial tight junctions of excitable tissue. As described above, theinvention is based, in part, on the discovery that EPO molecules can betransported from the luminal surface to the basement membrane surface ofendothelial cells of the capillaries of organs with endothelial celltight junctions, including, for example, the brain, retina, and testis.While not wishing to be bound by any particular theory, aftertranscytosis of EPO, EPO can interact with an EPO receptor on excitabletissue, such as, for example, neurons of the central nervous system, theperipheral nervous system, or heart tissue, and receptor binding caninitiate a signal transduction cascade resulting in the activation of agene expression program within the excitable tissue, resulting in theprotection of the cell from damage, such as by neurotoxins, hypoxia,etc. Thus, methods for protecting excitable tissue from injury orhypoxic stress, enhancing the function of excitable tissue, anddelivering compounds across tight junctions of excitable tissue aredescribed in detail hereinbelow.

5.2.1 Methods for Enhancing Excitable Tissue Function

In one aspect, the present invention is directed to a method forenhancing the function of excitable tissue by administration of an EPOmolecule capable of activating a gene expression program that enhancesexcitable tissue function. Enhancement of excitable tissue functionprovides enhancement of learning, associative learning, and memory.Various diseases and conditions are amenable to treatment using thismethod, and further, this method is useful for enhancing cognitivefunction in the absence of any condition or disease. These uses of thepresent invention are described in further detail below, and includeenhancement of learning and training in both human and non-humanmammals.

Conditions and diseases treatable by the methods of this aspect of thepresent invention include any condition or disease that can benefit fromenhancement of neuronal function. Examples of such disorders includedisorders of the central nervous system including, but not limited, tomood disorders, anxiety disorders, depression, autism, attention deficithyperactivity disorder, and cognitive dysfunction. Other non-limitingexamples of cognitive functions which can be enhanced using the methodsof the invention are described in Section 5.3.

In one embodiment, for example, an EPO molecule may be administered to asubject or patient who is suffering from a disorder resulting in loss ofcognitive functions, such, for example, as Alzheimer's Disease.

The ability of EPO to enhance cognitive functions can be tested inexperimental animals using any of the methods described herein, or anyother art-accepted learning or cognitive function model. As described inthe Examples presented in Sections 6 and 7, peripherally-administerederythropoietin was discovered to enhance learning and cognitive functionas demonstrated by several well established learning models in normalexperimental animals. Examples of such learning models are the Morriswater maze test, an example of which is given in Section 6 and theconditioned taste aversion (CTA) test, an example of which is given inSection 7. In one embodiment, for example, the conditioned tasteaversion (CTA), a very sensitive, well known, standard test is used totest an animal's cognitive function after administration of EPO. CTA isused to test an animal's ability for learning to associate illness witha novel stimuli, such as taste, such that the animals avoid the noveltaste upon subsequent re-exposure to the novel stimuli. CTA involves thebrain at a variety of cortical and subcortical levels. The associationwhich links ascending and descending information together producingaversive behavior can be either attenuated or strengthened by changesaffecting any of the interconnecting units. As a form of associativelearning, the strength of CTA is determined by a large number ofvariables including novelty of the oral stimulus (e.g., non-novelstimuli cannot be aversively conditioned), degree of “illness” produced(toxicity), number of repetitions (training), countering drives (such asthirst) to name a few. Although a wide variety of chemical and physicalagents can produce CTA in a dose-dependent manner, lithium chloridereliably produces malaise and anorexia. Like a naturally occurringillness, lithium produces a CTA by stimulating the pathways describedabove, including cytokine release.

Enhancement of excitable cell function, for example, cognitive function,offers numerous benefits to individuals in the educational and workenvironment, and to enhance the ability to train and educate non-humanmammals.

5.2.2 Methods for Protecting Excitable Tissue from Injury

In another embodiment, the present invention is directed toward a methodfor protecting a mammal from pathology resulting from injury toexcitable tissue. Protection is provided by administering to a mammal bya peripheral route of administration an amount of erythropoietineffective to protect the excitable tissue from injury. As is shown indetail in the example in Section 8, below, EPO administered in advanceof the toxin kainate is markedly neuroprotective in mice, raisingseizure threshold and preventing death. The neuroprotective effect EPOis large and is sustained. It is notable that the positive effects seenherein occur within too short of a time relative to the administrationof an EPO to be a result of an increase in hematocrit as a consequenceof the erythropoietic activity of EPO. Furthermore, as noted above, anembodiment of the present invention comprises an EPO which lacks theability to increase hematocrit.

In one embodiment, the present invention may be used advantageously bothin the acute and chronic prophylaxis and treatment of neurologicaldisorders, as described herein, and in enhanced cognitive function ofthe normal or the diseased brain. As noted above, damage and death ofneurons in the central nervous system is a serious and often lethaloccurrence responsible for a high degree of morbidity and mortality inthe population. Acute neurological damage may occur during or as aresult of seizures, convulsions, epilepsy, stroke, hemorrhage, centralnervous system injury, hypoxia, hypoglycemia, hypotension and brain orspinal cord trauma. The present invention provides for acuteadministration for the treatment of acute events.

In one embodiment, for example, the methods of the invention may be usedto protect a mammal from injury resulting from radiation damage to thebrain.

In another embodiment, a serious condition treatable or preventable inaccordance with the present invention is prophylaxis and treatment inutero of prenatal hypoxic conditions, post-birth treatment to protectthe brain from hypoxic injury sustained during birth, as well as insuffocation, drowning, and other conditions wherein the central nervoussystem is at risk for neurotoxic damage as a result of oxygendeprivation or exposure to other neurotoxic stimuli. As is well known,individuals who suffer from hypoxia during labor, or as a consequence ofnon-fatal hypoxic accidents or incidents may suffer a lifelongneurologic deficit. Hypoxia and/or cessation of cerebral blood flow,which may occur post-trauma or during surgical procedures, also carriesa risk of causing lifelong neurologic deficit.

Postoperative cognitive dysfunction, including deficits following theuse of a heart-lung machine, are also treatable by the methods providedherein. Furthermore, the present methods may be applied to the treatmentof hypoxia resulting from carbon monoxide poisoning or smoke inhalation.

In another embodiment, EPO is used to protect cardiac tissue from injurysustained during ischemia, infarction, inflammation, or trauma.

These are non-limiting examples of damage to excitable tissue treatablein accordance with the present invention. Acute and early treatment ofthese disorders may be carried out by mobile medical emergency healthcare professionals such that treatment may be started as soon assuspicion of potential for neurologic damage is ascertained. Risk ofneurologic damage induced by labor may be reduced by prophylacticallytreatment of the fetus before or during labor. These and other utilitiesand situations will be recognized by the skilled artisan.

5.2.3 Methods for Delivery of Compounds

The present invention is further directed to a method for facilitatingthe transport of a molecule across an endothelial cell barrier in amammal by administering a composition which comprises the particularmolecule in association with erythropoietin. As noted above, theinventors herein discovered the heretofore unexpected and surprisingactivity of peripherally-administered EPO on excitable tissue, such asnervous tissue in the central nervous system, the peripheral nervoussystem, or heart tissue, identifying EPO as a molecule capable ofcrossing tight junctions of such excitable tissue, such as theblood-brain barrier. As such, EPO is useful as a carrier for deliveringother molecules across the blood-brain and other similar barriers.

In one embodiment, EPO receptor binding molecules comprising moleculesconjugated to an EPO molecule, may be used to transport those moleculesacross the blood brain barrier. Such molecules can thereby piggyback onEPO for delivery across the BBB.

In another embodiment, an antibody or other binding partner to themolecule may be associated with EPO, or with an EPO receptor activitymodulator, thus associating the molecule to be transported bynoncovalent binding to the binding partner, which is further associatedwith the transportable EPO molecule. In another embodiment, EPOreceptor-binding molecules comprising antibodies to the EPO receptor areuseful for the method described here. Such antibodies provide atransport carrier on which other molecules may hitchhike, much in thesame fashion that antibodies to the transferrin receptor have been usedto gain access across the blood-brain (Pardridge et al., 1991, Selectivetransport of an antitransferrin receptor antibody through theblood-brain barrier in vivo. J. Pharmacol. Exp. Therap. 27: 66).

The skilled artisan will be aware of various means for associatingmolecules with EPO and the other agents described above, by covalent,non-covalent, and other means; furthermore, evaluation of the efficacyof the composition may be readily determined in an experimental system.Association of molecules with EPO and analogs may be achieved by anynumber of means, including labile, covalent binding, cross-linking, etc.In one embodiment, for example, the association between the molecule tobe transported across the barrier and the erythropoietin may be a labilecovalent bond, in which case the molecule is released from associationwith the EPO after crossing the barrier. In one embodiment,biotin/avidin interactions may be employed. In another embodiment, asmentioned above, a hybrid molecule may be prepared by recombinant orsynthetic means, for example, which includes both the domain of themolecule with desired pharmacological activity and the domainresponsible for EPO receptor activity modulation.

A molecule may be conjugated to an EPO or EPO receptor activitymodulator through a polyfunctional molecule, i.e., a polyfunctionalcrosslinker. As used herein, the term “polyfunctional molecule”encompasses molecules having one functional group that can react morethan one time in succession, such as formaldehyde, as well as moleculeswith more than one reactive group. As used herein, the term “reactivegroup” refers to a functional group on the crosslinker that reacts witha functional group on a molecule (e.g., peptide, protein, carbohydrate,nucleic acid, particularly a hormone, antibiotic, or anti-cancer agentto be delivered across an endothelial cell barrier) so as to form acovalent bond between the cross-linker and that molecule. The term“functional group” retains its standard meaning in organic chemistry.The polyfunctional molecules which can be used are preferablybiocompatible linkers, i.e., they are noncarcinogenic, nontoxic, andsubstantially non-immunogenic in viva. Polyfunctional cross-linkers suchas those known in the art and described herein can be readily tested inanimal models to determine their biocompatibility. The polyfunctionalmolecule is preferably bifunctional. As used herein, the term“bifunctional molecule” refers to a molecule with two reactive groups.The bifunctional molecule may be heterobifunctional or homobifunctional.A heterobifunctional cross-linker allows for vectorial conjugation. Itis particularly preferred for the polyfunctional molecule to besufficiently soluble in water for the cross-linking reactions to occurin aqueous solutions such as in aqueous solutions buffered at pH 6 to 8,and for the resulting conjugate to remain water soluble for moreeffective bio-distribution. Typically, the polyfunctional moleculecovalently bonds with an amino or a sulfhydryl functional group.However, polyfunctional molecules reactive with other functional groups,such as carboxylic acids or hydroxyl groups, are contemplated in thepresent invention.

The homobifunctional molecules have at least two reactive functionalgroups, which are the same. The reactive functional groups on ahomobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde and subaraldehyde. The use ofglutaraldehyde as a cross-linking agent was disclosed by Poznansky etal., Science 223, 1304-1306 (1984). Homobifunctional molecules having atleast two active ester units include esters of dicarboxylic acids andN-hydroxysuccinimide. Some examples of such N-succinimidyl estersinclude disuccinimidyl suberate and dithio-bis-(succinimidylpropionate), and their soluble bis-sulfonic acid and bis-sulfonate saltssuch as their sodium and potassium salts. These homobifunctionalreagents are available from Pierce, Rockford, Ill.

The heterobifunctional molecules have at least two different reactivegroups. The reactive groups react with different functional groups,e.g., present on the EPO and the molecule. These two differentfunctional groups that react with the reactive group on theheterobifunctional cross-linker are usually an amino group, e.g., theepsilon amino group of lysine; a sulfhydryl group, e.g., the thiol groupof cysteine; a carboxylic acid, e.g., the carboxylate on aspartic acid;or a hydroxyl group, e.g., the hydroxyl group on serine.

When a reactive group of a heterobifunctional molecule forms a covalentbond with an amino group, the covalent bond will usually be an amido orimido bond. The reactive group that forms a covalent bond with an aminogroup may, for example, be an activated carboxylate group, ahalocarbonyl group, or an ester group. The preferred halocarbonyl groupis a chlorocarbonyl group. The ester groups are preferably reactiveester groups such as, for example, an N-hydroxy-succinimide ester group.

The other functional group typically is either a thiol group, a groupcapable of being converted into a thiol group, or a group that forms acovalent bond with a thiol group. The covalent bond will usually be athioether bond or a disulfide. The reactive group that forms a covalentbond with a thiol group may, for example, be a double bond that reactswith thiol groups or an activated disulfide. A reactive group containinga double bond capable of reacting with a thiol group is the maleimidogroup, although others, such as acrylonitrile, are also possible. Areactive disulfide group may, for example, be a 2-pyridyldithio group ora 5,5′-dithio-bis-(2-nitrobenzoic acid) group. Some examples ofheterobifunctional reagents containing reactive disulfide bonds includeN-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson et al., 1978,Biochem J., 173:723-737), sodium5-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl(2-pyridyldithio) toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidylm-maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate saltof succinimidyl m-maleimidobenzoate is preferred. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce.

The need for the above-described conjugated to be reversible or labilemay be readily determined by the skilled artisan. A conjugate may betested in vitro for both the EPO receptor activity modulation activity,and for the desirable pharmacological activity. If the conjugate retainsboth properties, its suitability may then be tested in vivo. If theconjugated molecule requires separation from the EPO for activity, alabile bond or reversible association with EPO will be preferable. Theliability characteristics may also be tested using standard in vitroprocedures before in vivo testing.

Additional information regarding how to make and use these as well asother polyfunctional reagents may be obtained from the followingpublications or others available in the art: Carlsson et al., 1978,Biochem. J. 173:723-737; Cumber et al., 1985, Methods in Enzymology112:207-224; Jue et al., 1978, Biochem. 17:5399-5405; Sun et al., 1974,Biochem. 13:2334-2340; Blattler et al., 1985, Biochem. 24:1517-152; Liuet al., 1979, Biochem. 18:690-697; Youle and Neville, 1980, Proc. Natl.Acad. Sci. U.S.A. 77:5483-5486; Lerner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:3403-3407; Jung and Moroi, 1983, Biochem. Biophys. Acta761:162; Caulfield et al., 1984, Biochem. 81:7772-7776; Staros, J. V.,1982, Biochem. 21:3950-3955; Yoshitake et al., 1979, Eur. J. Biochem.101:395-399; Yoshitake et al., 1982, J. Biochem. 92:1413-1424; Pilch andCzech, 1979, J. Biol. Chem. 254:3375-3381; Novick et al., 1987, J. Biol.Chem. 262:8483-8487; Lomant and Fairbanks, 1976, J. Mol. Biol.104:243-261; Hamada and Tsuruo, 1987, Anal. Biochem. 160:483-488; andHashida, 1984, J. Applied Biochem. 6:56-63. Additionally, methods ofcross-linking are reviewed by Means and Feeney, 1990, Bioconjugate Chem.1:2-12. Barriers which are crossed by the above-described methods andcompositions of the present invention include but are not limited to theblood-brain barrier, the blood-eye barrier, the blood-testes barrier,the blood-ovary barrier, and the blood-placenta barrier.

Candidate molecules for transport across an endothelial cell barrierinclude, for example, hormones such as growth hormone, neurotrophicfactors, antibiotics or antifungals such as those normally excluded fromthe brain and other barriered organs, peptide radiopharmaceuticals,antisense drugs, antibodies against biologically-active agents,pharmaceuticals, and anti-cancer agents. Non-limiting examples of suchmolecules include growth hormone, nerve growth factor (NGF),brain-derived neurotrophic factor (BNF), ciliary neurotrophic factor(CTF.), basic fibroblast growth factor (bFGF), transforming growthfactor β1 (TGFβ1), transforming growth factor β2 (TGFβ2), transforminggrowth factor β3 (TGFβ3), interleukin 1, interleukin 2, interleukin 3,and interleukin 6, AZT, antibodies against tumor necrosis factor, andimmunosuppressive agents such as cyclosporin.

In another embodiment, recombinant chimeric toxin molecules comprisingEPO can be used for therapeutic delivery of toxins to treat a viraldisorder or proliferative disorder, such as cancer. Compounds that couldbe fused to EPO to construct a chimeric toxin suitable for thisembodiment include, but are not limited to, toxic substances, such aspseudomonas exotoxin, diphtheria toxin, and ricin, among others.

5.3 Target Conditions

As described above, the EPO compositions and methods for their useprovided herein can be used to treat and prevent conditions arising fromhypoxic conditions, which adversely affect excitable tissues, such asexcitable tissues in the central nervous system tissue, peripheralnervous system tissue, or cardiac tissue such as, for example, brain,heart, or retina. Therefore, the invention can be used to treat orprevent damage to excitable tissue resulting from hypoxic conditions ina variety of conditions and circumstances. Non-limiting examples of suchconditions and circumstances are provided hereinbelow.

In the example of the protection of neuronal tissue pathologiestreatable in accordance with the present invention, such pathologiesinclude those which result from reduced oxygenation of neuronal tissues.Any condition which reduces the availability of oxygen to neuronaltissue, resulting in stress, damage, and finally, neuronal cell death,can be treated by the methods of the present invention. Generallyreferred to as hypoxia and/or ischemia, these conditions arise from orinclude, but are not limited to stroke, vascular occlusion, prenatal orpostnatal oxygen deprivation, suffocation, choking, near drowning,carbon monoxide poisoning, smoke inhalation, trauma, including surgeryand radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic obstructivepulmonary disease, emphysema, adult respiratory distress syndrome,hypotensive shock, septic shock, anaphylactic shock, insulin shock,sickle cell crisis, cardiac arrest, dysrhythmia, and nitrogen narcosis.

In one embodiment, for example, EPO may be administered to preventinjury or tissue damage resulting from risk of injury or tissue damageduring surgical procedures, such as, for example, tumor resection oraneurysm repair.

Other pathologies caused by or resulting from hypoglycemia which aretreatable by the methods described herein include insulin overdose, alsoreferred to as iatrogenic hyperinsulinemia, insulinoma, growth hormonedeficiency, hypocortisolism, drug overdose, and certain tumors.

Other pathologies resulting from excitable neuronal tissue damageinclude seizure disorders, such as epilepsy, convulsions, or chronicseizure disorders. Other treatable conditions and diseases includediseases such as stroke, multiple sclerosis, hypotension, cardiacarrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, brainor spinal cord trauma, AIDS dementia, age-related loss of cognitivefunction, memory loss, amyotrophic lateral sclerosis, seizure disorders,alcoholism, retinal ischemia, optic nerve damage resulting fromglaucoma, and neuronal loss.

The methods of the invention may be used to treat conditions of, anddamage to, retinal tissue. Such disorders include, but are not limitedto macular degeneration, retinal detachment, retinitis pigmentosa,arteriosclerotic retinopathy, hypertensive retinopathy, retinal arteryblockage, retinal vein blockage, hypotension, and diabetic retinopathy.

In another embodiment, the methods of the invention may be used toprotect or treat injury resulting from radiation damage to excitabletissue.

A further utility of the methods of the present invention is in thetreatment of neurotoxin poisoning, such as domoic acid shellfishpoisoning, neurolathyrism, and Guam disease, amyotrophic lateralsclerosis, and Parkinson's disease.

As mentioned above, the present invention is also directed to a methodfor enhancing excitable tissue function in a mammal by peripheraladministration of erythropoietin. Various diseases and conditions areamenable to treatment using this method, and further, this method isuseful for enhancing cognitive function in the absence of any conditionor disease. These uses of the present invention are described in furtherdetail below, and include enhancement of learning and training in bothhuman and non-human mammals.

Conditions and diseases treatable by the methods of this aspect of thepresent invention directed to the central nervous system include but arenot limited to mood disorders, anxiety disorders, depression, autism,attention deficit hyperactivity disorder, and cognitive dysfunction.These conditions benefit from enhancement of neuronal function. Otherdisorders treatable in accordance with the teachings of the presentinvention include sleep disruption, for example, sleep apnea andtravel-related disorders; subarachnoid and aneurysmal bleeds,hypotensive shock, concussive injury, septic shock, anaphylactic shock,and sequelae of various encephalitides and meningitides, for example,connective tissue disease-related cerebritides such as lupus. Other usesinclude prevention of or protection from poisoning by neurotoxins, suchas domoic acid shellfish poisoning, neurolathyrism, and Guam disease,amyotrophic lateral sclerosis, Parkinson's disease; postoperativetreatment for embolic or ischemic injury; whole brain irradiation;sickle cell crisis; and eclampsia.

A further group of conditions treatable by the methods of the presentinvention include mitochondrial dysfunction, of either an hereditary oracquired nature, which are the cause of a variety of neurologicaldiseases typified by neuronal injury and death. For example, Leighdisease (subacute necrotizing encephalopathy) is characterized byprogressive visual loss and encephalopathy, due to neuronal drop out,and myopathy. In these cases, defective mitochondrial metabolism failsto supply enough high energy substrates to fuel the metabolism ofexcitable cells. An EPO receptor activity modulator optimizes failingfunction in a variety of mitochondrial diseases.

As mentioned above, hypoxic conditions adversely affect excitabletissues. The excitable tissues include, but are not limited to, centralnervous system tissue, peripheral nervous system tissue, and hearttissue. In addition to the conditions described above, the methods ofthe present invention are useful in the treatment of inhalationpoisoning such as carbon monoxide and smoke inhalation, severe asthma,adult respiratory distress syndrome, and choking and near drowning.Further conditions which create hypoxic conditions or by other meansinduce excitable tissue damage include hypoglycemia that may occur ininappropriate dosing of insulin, or with insulin-producing neoplasms(insulinoma).

Various neuropsychologic disorders which are believed to originate fromexcitable tissue damage are treatable by the instant methods. Chronicdisorders in which neuronal damage may be involved and for whichtreatment by the present invention is provided include disordersrelating to the central nervous system and/or peripheral nervous systemincluding age-related loss of cognitive function and senile dementia,chronic seizure disorders, Alzheimer's disease, Parkinson's disease,dementia, memory loss, amyotrophic lateral sclerosis, multiplesclerosis, tuberous sclerosis, Wilson's Disease, cerebral andprogressive supranuclear palsy, Guam disease, Lewy body dementia, priondiseases, such as spongiform encephalopathies, e.g., Creutzfeldt-Jakobdisease, Huntington's disease, myotonic dystrophy, Freidrich's ataxiaand other ataxias, as well as Gilles de la Tourette's syndrome, seizuredisorders such as epilepsy and chronic seizure disorder, stroke, brainor spinal cord trauma, AIDS dementia, alcoholism, autism, retinalischemia, glaucoma, autonomic function disorders such as hypertensionand sleep disorders, and neuropsychiatric disorders that include, butare not limited to schizophrenia, schizoaffective disorder, attentiondeficit disorder, dysthyrnic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, panic disorder, as well as unipolar and bipolar affectivedisorders. Additional neuropsychiatric and neurodegenerative disordersinclude, for example, those listed in the American PsychiatricAssociation's Diagnostic and Statistical manual of Mental Disorders(DSM), the most current version of which is incorporated herein byreference in its entirety.

In another embodiment, recombinant chimeric toxin molecules comprisingEPO can be used for therapeutic delivery of toxins to treat aproliferative disorder, such as cancer, or viral disorder, such assubacute sclerosing panencephalitis.

5.4 Pharmaceutical Preparations and Administration

According to the invention, EPO, its analogues, mimetics, erythropoietinfragments, hybrid erythropoietin molecules, erythropoietinreceptor-binding molecules, erythropoietin agonists, renalerythropoietin, brain erythropoietin, muteins thereof, and congenersthereof, may be introduced parenterally, transmucosally, e.g., orally,nasally, rectally, intravaginally, sublingually, submucosally ortransdermally. Preferably, administration is parenteral, e.g., viaintravenous or intraperitoneal injection, and also including, but is notlimited to, intra-arterial, intramuscular, intradermal and subcutaneousadministration. The preferred route of administration of small moleculeEPO mimetics is by the oral route.

A subject in whom peripheral administration of EPO is an effectivetherapeutic regiment is preferably a human, but can be any animal,preferably a mammal. Thus, as can be readily appreciated by one ofordinary skill in the art, the methods and pharmaceutical compositionsof the present invention are particularly suited to administration toany animal, particularly a mammal, and including, but by no meanslimited to, domestic animals, such as feline or canine subjects, farmanimals, such as but not limited to bovine, equine, caprine, ovine, andporcine subjects, wild animals (whether in the wild or in a zoologicalgarden), research animals, such as mice, rats, rabbits, goats, sheep,pigs, dogs, cats, etc. As noted above, domesticated animals, includingpets and work animals, are candidates for both the neuroprotectivebenefits of the present invention, as well as the enhancement ofcognitive function. Neurological damage arising from hypoxia, and wellas acute and chronic disorders including epilepsy, are common among suchanimals, and thus are candidates for treatment. Also as noted above,cognitive enhancement in non-human animals is a benefit of the presentinvention, in that learning, training, and retention of learned behaviormay be enhanced, reinforced, and maintained using the teachings of thepresent invention. As such, the expense and psychological strain to thepet owner is reduced. For example, the time required for training dogsand other domestic animals is reduced. Furthermore, wild animalstypically difficult to train may be better candidates for training bythe methods of the present invention.

5.4.1 Formulation and Effective Dose

The present invention also provides pharmaceutical compositions.Pharmaceutical compositions comprising EPO and EPO receptor activitymodulators can be administered to a patient at therapeutically effectivedoses to protect excitable tissue from damage, enhance the function ofexcitable tissue, or to deliver a compound to excitable tissue. TheApplicants have discovered that an elevated dose of EPO is preferred tomodulate excitable tissue, and to protect against injury thereto.

Selection of the preferred effective dose will be determined by askilled artisan based upon considering several factors which will beknown to one of ordinary skill in the art. Such factors include theparticular form of erythropoietin, and its pharmacokinetic parameterssuch as bioavailability, metabolism, half-life, etc., which will havebeen established during the usual development procedures typicallyemployed in obtaining regulatory approval for a pharmaceutical compound.Further factors in considering the dose include the condition or diseaseto be treated or the benefit to be achieved in a normal individual, thebody mass of the patient, the route of administration, whetheradministration is acute or chronic, concomitant medications, and otherfactors well known to affect the efficacy of administered pharmaceuticalagents. Thus the precise dosage should be decided according to thejudgment of the practitioner and each patient's circumstances, e.g.,depending upon the condition and the immune status of the individualpatient, according to standard clinical techniques.

In one embodiment, the invention provides a pharmaceutical compositionin dosage unit form adapted for modulation of excitable tissue,enhancement of cognitive function or delivery of compounds acrossendothelial tight junctions which comprises, per dosage unit, aneffective non-toxic amount within the range from about 50,000 to 500,000Units, 60,000 to 500,000 Units, 70,000 to 500,000 Units, 80,000 to500,000 Units, 90,000 to 500,000 Units, 100,000 to 500,000 Units,150,000 to 500,000 Units, 200,000 to 500,000 Units, 250,000 to 500,000Units, 300,000 to 500,000 Units, 350,000 to 500,000 Units, 400,000 to500,000 Units, or 450,000 to 500,000 Units of EPO, an EPO receptoractivity modulator, or an EPO-activated receptor modulator and apharmaceutically acceptable carrier. In a preferred embodiment, theeffective non-toxic amount of EPO is within the range from about 50,000to 500,000 Units.

In one embodiment, such a pharmaceutical composition of EPO may beadministered systemically to protect excitable tissue from damage,enhance the function of excitable tissue, or to deliver a compound toexcitable tissue. Such administration may be parenterally,transmucosally, e.g., orally, nasally, rectally, intravaginally,sublingually, submucosally or transdermally. Preferably, administrationis parenteral, e.g., via intravenous or intraperitoneal injection, andalso including, but is not limited to, intra-arterial, intramuscular,intradermal and subcutaneous administration.

In a preferred embodiment, EPO may be administered systemically at adosage between 2000-10000 Units/kg body weight, preferably about2000-5000 Units/kg-body weight, most preferably 5000 Units/kg-bodyweight, per administration. This effective dose should be sufficient toachieve serum levels of EPO greater than about 10,000, 15,000, or 20,000mU/ml of serum after EPO administration. Such serum levels may beachieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hourspost-administration. Such dosages may be repeated as necessary. Forexample, administration may be repeated daily, as long as clinicallynecessary, or after an appropriate interval, e.g., every 1 to 12 weeks,preferably, every 3 to 8 weeks. In one embodiment, the effective amountof EPO and a pharmaceutically acceptable carrier may be packaged in asingle dose vial or other container. In one embodiment, an EPO isnonerythropoietic, i.e., it is capable of exerting the activitiesdescribed herein but not causing an increase in hemoglobin concentrationor hematocrit. In another embodiment, an EPO is given at a dose greaterthan that necessary to maximally stimulate erythropoiesis.

The pharmaceutical compositions of the invention may comprise atherapeutically effective amount of a compound, and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as saline solutions in water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. A saline solution is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. The compounds of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin. Such compositions will contain atherapeutically effective amount of the compound, preferably in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. The formulation shouldsuit the mode of administration.

Pharmaceutical compositions adapted for oral administration may beprovided as capsules or tablets; as powders or granules; as solutions,syrups or suspensions (in aqueous or non-aqueous liquids); as ediblefoams or whips; or as emulsions. Tablets or hard gelatine capsules maycomprise lactose, starch or derivatives thereof, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, stearic acid or saltsthereof. Soft gelatine capsules may comprise vegetable oils, waxes,fats, semi-solid, or liquid polyols etc. Solutions and syrups maycomprise water, polyols and sugars.

An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract (e.g., glycerylmonostearate or glyceryl distearate may be used). Thus, the sustainedrelease of an active agent may be achieved over many hours and, ifnecessary, the active agent can be protected from being degraded withinthe stomach. Pharmaceutical compositions for oral administration may beformulated to facilitate release of an active agent at a particulargastrointestinal location due to specific pH or enzymatic conditions.Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.Pharmaceutical compositions adapted for topical administration may beprovided as ointments, creams, suspensions, lotions, powders, solutions,pastes, gels, sprays, aerosols or oils. For topical administration tothe skin, mouth, eye or other external tissues a topical ointment orcream is preferably used. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water base or a water-in-oil base. Pharmaceuticalcompositions adapted for topical administration to the eye include eyedrops. In these compositions, the active ingredient can be dissolved orsuspended in a suitable carrier, e.g., in an aqueous solvent.Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions adapted for nasal administration maycomprise solid carriers such as powders (preferably having a particlesize in the range of 20 to 500 microns). Powders can be administered inthe manner in which snuff is taken, i.e., by rapid inhalation throughthe nose from a container of powder held close to the nose.Alternatively, compositions adopted for nasal administration maycomprise liquid carriers, e.g., nasal sprays or nasal drops. Thesecompositions may comprise aqueous or oil solutions of the activeingredient. Compositions for administration by inhalation may besupplied in specially adapted devices including, but not limited to,pressurized aerosols, nebulizers or insufflators, which can beconstructed so as to provide predetermined dosages of the activeingredient. In a preferred embodiment, pharmaceutical compositions ofthe invention are administered via the nasal cavity to the lungs.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided as pessaries, tampons,creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injectable solutions orsuspensions, which may contain antioxidants, buffers, bacteriostats andsolutes that render the compositions substantially isotonic with theblood of an intended recipient. Other components that may be present insuch compositions include water, alcohols, polyols, glycerine andvegetable oils, for example. Compositions adapted for parenteraladministration may be presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of asterile liquid carrier, e.g., sterile saline solution for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.4.2 Methods of Administration

The present invention provides compositions and methods for peripheraladministration of EPO to enhance function or protect excitable tissues,and to deliver compounds to such tissues. As noted above, the presentinvention is based, in part, on the discovery that peripherallyadministered EPO has direct neuroprotective or neuroenhancementproperties in excitable tissue, such as tissue of the central nervoussystem, peripheral nervous system, or heart tissue. Excitable tissue, asused herein, includes, but is not limited to, neuronal tissue of thecentral and peripheral nervous systems, and cardiac tissue. This sectiondescribes such compounds, and their methods for their of administration.

The present invention provides for administration of EPO and EPOreceptor activity modulators by routes of administration other thandirectly into the central nervous system, and the terms “peripheral” and“systemic” subsumes these various routes. Peripheral administrationincludes oral or parenteral administration, such as intravenous,intraarterial, subcutaneous, intramuscular, intraperitoneal, rectal,submucosal or intradermal administration. Other routes are useful forthe administration of the agents described herein. Both acute andchronic administration are provided herein.

In one embodiment, for example, EPO can be delivered in acontrolled-release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRCCrit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507;Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment,the compound can be delivered in a vesicle, in particular a liposome(see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes inthe Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); WO 91/04014; U.S. Pat. No.4,704,355; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). Inanother embodiment, polymeric materials can be used [see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Press:Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley: New York (1984);Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1953;see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, pp. 115-138 inMedical Applications of Controlled Release, vol. 2, supra, 1984). Othercontrolled release systems are discussed in the review by Langer (1990,Science 249:1527-1533).

In another embodiment, EPO, as properly formulated, can be administeredby nasal, oral, rectal, vaginal, or sublingual administration.

In a specific embodiment, it may be desirable to administer the EPOcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

As is described hereinbelow, the studies that were performed by theinventors herein are standard, universally-accepted tests in animalmodels predictive of prophylactic and therapeutic benefit.

6. EXAMPLE 1 Peripherally Administered EPO Enhances Cognitive Function

In this Example, a spatial navigation experiment, known as the MorrisWater Maze test, demonstrates EPO-induced enhancement of cognitivefunction in mice. In this test, a small transparent platform is placedin one quadrant of a swimming pool with opaque water. Mice placed intothis swimming pool must swim until they reach the resting platform belowthe surface, which is invisible to the swimming mice. The test consistsof measuring the time the animals take to get to the platform (i.e., thelength of time they spend swimming). On successive trials, the time eachmouse takes to reach the platform will decrease as a function of themlearning its location. This type of learning experiment involves thehippocampus, as hippocampal lesions prevent learning in this test.

Experiments were carried out in a circular black pool, 150 cm indiameter. Four points were arbitrarily assigned: north, south, east andwest. Distinctive visual cues were applied to each of these fourquadrants: e.g., flashing lights, bright tape applied in squares etc.,to orient the mice in the pool. A platform was arbitrarily placed in onequadrant. A trial consisted of placing the animal head-first in onequadrant of the pool and releasing it. The trial length was 90 secondstotal. If the animal did not make it to the platform, she was placed onit for an additional 15 seconds. The subjects were rested for an hour,then placed in another quadrant for testing. All 4 quadrants were usedover the course of a day's trials, and the animals were tested on 12successive days (i.e., a total of 48 trials).

The experiment itself consisted of injecting each mice with 5000 U/kgrecombinant human EPO (sold under the tradename of PROCRIT,Ortho-Biotech, Inc.) by intraperitoneal injection, 4 hours before thebeginning of the day's testing, each day for the 12 trial days. Controlanimals were sham-injected with saline.

Learning was measured by measuring the length of time each mouse spenton the platform. Shown in FIG. 1A, the results are plotted as the timespent on the platform by the EPO-treated group and the sham group. Asthe results indicate, both groups of animals spent more time on theplatform, i.e. they learned to reach the platform faster, on eachsuccessive trial day, but the EPO-treated animals did so faster than thesham group. Thus EPO-treated animals have a much faster “learning curve”than the sham group. When results were expressed as the differencebetween the EPO-treated and the sham-treated groups, and the results ofthe EPO and the sham-treated group were compared, the regression line(R²=0.88) shows a slope (0.68) significantly different from a slope of1, markedly in favor of the EPO group (FIG. 1B).

7. EXAMPLE 2 Peripherally Administered EPO Strengthens a LearnedConditioned Taste Aversion

The Conditioned Taste Aversion (CTA) test performed in this Exampledemonstrates that EPO dramatically affects the ability of mice toremember, and learn to avoid, an unpleasant taste sensation, in thiscase an illness-provoking substance. In this example, lithium chlorideis used to produce CTA, because lithium chloride reliably producesmalaise and anorexia in a dose-dependent manner. Like a naturallyoccurring illness, lithium produces a CTA by stimulating the pathwaysdescribed above, including cytokine release.

Female Balb/c mice were trained to limit their total daily water intaketo a single minute drinking period per day, and learned to drink enoughwater during this period to remain at equilibrium. Animals were dividedinto groups and administered either a sham control (saline) or EPO (5000U/kg), injected intraperitoneally (IP), 4 hours before presentation of anovel saccharin-vanilla liquid. Immediately after finishing drinking thesweet liquid, animals received either saline or an illness-producingdose of lithium (20 mg/kg of a 0.15 M LiCl, delivered IP). Thereafter,three groups of animals were followed. The first group (control) did notreceive lithium after drinking. The second group received both lithiumand EPO. The third group (sham) received saline (without EPO) andlithium.

Conditioned Taste Aversion was measured by measuring the reduction indrinking upon subsequent exposure to the illness-producing solution,novel saccharin-vanilla liquid. After a 5-day recovery from the lithiumor sham treatment, water-deprived animals were presented again with thesame novel saccharin-vanilla liquid. The results plotted for groups 2and 3, compared to 1 (control) are shown in FIG. 2A. Day 2 representsthe baseline consumption of water after habituation to the test cage. OnDay 3, animals received an intraperitoneal injection of either saline orEPO (5000 U/kg) 4 hrs before presentation of the novel saccharin-vanillafluid, followed by treatment with lithium or a sham saline (arrow). Thistreatment resulted in a small decrease in fluid consumption in allgroups on Day 3, a previously documented adverse effect of the injectionand novelty of the fluid. After recovery, the first test for theestablishment of a CTA showed no decrease in consumption for controls.However, animals having received lithium demonstrated a virtuallycomplete aversion to the fluid, in spite of being water deprived (Day4). Continued deprivation of water eventually produced an extinction ofthe CTA (Days 5 to 9), but was characterized by a markedly delayedrecovery by the animals which had received EPO, as shown by the filledcircles in FIG. 2A.

The robustness of the CTA established herein is better appreciated byconsidering the degree of water deficit present on each test day, as theEPO-treated animals tolerated a water deficit approximately twice thatof sham-injected subjects (FIG. 2B). In spite of the markedlyaccentuated CTA demonstrated by the EPO group, the animals in this groupmore readily approached the drinking tube compared to the sham group, asshown in FIG. 2C. The strength of the CTA was demonstrated by a repeatinjection of lithium alone (without EPO) which produced an attenuatedCTA which was greater in the EPO group (FIG. 2A Day 10). These data showthat EPO pre-treatment is associated with a markedly potentiated CTAproduced by lithium.

8. EXAMPLE 3 Peripherally Adminstered EPO Protects Brain from anExcitotoxin

This Example demonstrates that EPO crosses the blood brain barrier andhas a neuroprotective effect in mice treated with the neurotoxinkainate. Many compounds exist in nature which exhibit toxicityspecifically for neurons. These molecules typically interact withendogenous receptors for the amino acid transmitter glutamate,subsequently causing excessive stimulation and neuronal injury. One ofthese, kainate, a substance widely used to study neuronal injury due toexcitotoxicity, is an analogue of glutamate. Kainate is a potentneurotoxin which specifically destroys neurons, particularly thoselocated in regions with a high density of kainate receptors, such as thehippocampus, and induces seizures, brain injury, and death.

The following neurotoxicity studies were performed with mice usingkainate. This model is used to assess the protective benefit oftreatments for conditions such as temporal lobe epilepsy. Parenteralinjections in experimental animals such as rats and mice elicit partial(limbic) seizures in a dose-dependent manner, which then may generalizeand cause death. The experiments presented in this section wereperformed to test whether peripherally-administered EPO crosses theblood brain barrier, and if so, whether EPO has an effect on neuronalenergy balance, and specifically, it has neuroprotective effects againstkainate.

To this end, female Balb/c mice (weighing on average 15-20 gm) werepretested with 5000 U/kg of recombinant human erythropoietin (rhEPO;sold under the mark PROCRIT, Ortho-Biotech, Inc.) or saline (sham) givenintraperitoneally at specific time points before, at or after receivingkainate (Sigma Chemical), also IP, at specific concentrations(mass/kg-body weight). Subjects were then monitored and graded for thedevelopment of seizure activity at 20 minutes after receiving kainate.Each trial was terminated 60 minutes after the kainate dose. As shown inFIG. 3A, EPO pretreatment dramatically reduces seizure severity anddelays the onset of status epilepticus in mice treated with kainate. Thecomparison between EPO- and sham-treated animals demonstrates asignificantly lower death rate in animals receiving kainate dosages inthe 20-30 mg/kg range, indicating neuroprotection afforded bypretreatment with EPO. The numbers in parentheses under each columnindicate the number of animals exposed to each kainate dose.

The dose-dependency of EPO in providing neuroprotection from kainate isshown in FIG. 3B. Mice were administered EPO (5000 U/kg; IP daily for upto five days). The neuroprotective effect of each dose of EPO wasassessed by determining survival after administration of kainate (20mg/kg), which produces an approximate 50% mortality for control animals(no EPO; see FIG. 3A). Columns indicate improvement in survival ofEPO-treated subjects, compared to sham-injected animals. As shown inFIG. 3B, neuroprotection increases with additional dosages of 5000 U/kgof EPO.

The neuroprotection provided by EPO is characterized by a delayed onset,characteristic of the activation of a gene expression program. FIG. 3Cshows the EPO-related delay (in minutes) in death from seizures of asingle dose of EPO given at the time of kainate administration (20mg/kg) does not provide any immediate protection, whereas EPO given 24hours before kainate improves the latency and severity of seizures andtime to death. This effect lasts for up to 7 days.

9. EXAMPLE 4 Peripherally Administered EPO Protects Brain from DamageDue to Ischemia

Previous in vivo studies using a global reperfusion model in the gerbil,have indicated that stopping blood flow to the brain results in celldeath in the brain, and that EPO injected directly into the cerebralventricles protects the brain from such cell death (Sakanaka et al.,1998, Proc. Natl. Acad. Sci. U.S.A. 95:4635). The experiments presentedin this Example, for the first time, show that EPO deliveredperipherally protects the neural cell death in vivo in an animal modelof ischemia.

The following experiment was performed using the middle cerebral arteryocclusion model, an art-accepted model of ischemic focal stroke. In theprotocol, male rats (250 gm of body weight) were anesthetized withphenobarbital, and maintained at 37° C. The carotid arteries werevisualized, and the ipsalateral carotid artery permanently occluded. Theipsalateral middle cerebral artery (MCA) was visualized and cauterizedat its origin. The contralateral artery was occluded by clamping for 1hour. Animals were sacrificed 24 hours later, and the brain removed andsectioned into 1 mm serial sections. Viable tissue was visualized by insitu triphenyltetrazolium reduction to visualize live tissue fromnecrotic regions. The ischemic core, and the surrounding penumbra,undergoes cell death.

Using this MCA model, EPO was administered by parenteral injections atvarious times before and immediately after the injury, and the volume ofthe injury was quantified by computer-assisted image analysis. Theresults of this analysis, shown in FIG. 4A, indicated the effect oftreatment with EPO at the following times after the stroke: 24 hoursbefore the stroke, at the time of the stroke, and 3, 6, and 9 hoursafter the stroke. As shown in FIG. 4A, EPO protects tissue from necroticinjury when administered up to 6 hours post stroke.

Interestingly and in contrast, a 17-mer derived from EPO, which had beenpreviously reported to have neurotropic activity, promoting neuritegrowth in vitro and nerve cell myelination ex vivo (Campana et al.,1998, Int. J. Mol. Med. 1:235-41; U.S. Pat. No. 5,700,909 issued Dec.23, 1997), had no effect in protection against injury in this system(FIG. 4B, “17-mer”). Thus, this model, as well as the other methods forassaying the effect of EPO on excitable tissue function provided by thepresent invention, can be used to identify EPO and EPO receptor activitymodulators which can be used to modulate excitable tissue function, suchas protection from injury, or enhancement of learning and cognition.

10. EXAMPLE 5 Peripherally Administered EPO Protects Brain from BluntTrauma

In a model of mechanical trauma, the cortical impact model, pretreatmentwith systemically-administered EPO protects mouse brain from blunttrauma. To induce trauma a pneumatically-driven piston (ClippardValves), 3 mm in diameter which can precisely deliver a blow to theskull was employed. Each mouse was anesthetized and placed securely in asterotaxic device, to prevent the head from moving. A scalp incision wasmade in order to determine the location of the bregma, which is thereference point with which the piston was initially positioned. Thepiston was then adjusted by moving it 2 mm caudal and 2 mm ventral tobregma and the impact made by use of a precise pulse of nitrogen. Thisdevice allows for a precise selection of piston velocity (4 m/s) andimpact displacement (2 mm).

Mice were treated with EPO (5000 U/kg) 24 hours before, at the time ofinjury, 3, 6, or 9 hours later and continued as daily dosages. Mice weresacrificed 10 days after the procedure, and the brains subsequentlyexamined and the volume of brain necrosis determined. In sham-treatedmice, a large area of necrosis was observed (FIG. 5), and with abundantinfiltration of monocytes. In contrast, animals are protected from suchdamage, and few mononuclear cells were detected in the area of injury,when animals are pre-treated with EPO or given EPO up to 3 hours afterinjury.

11. EXAMPLE 6 Peripherally Administered EPO Protects Myocardium fromIschemic Injury

This Example demonstrates the effect of EPO in protection of hearttissue against hypoxic injury. To accomplish this, rats were pretestedwith EPO (5000 U/kg) 24 hours before the procedure performed as perLatini et al., (1999, J. Cardiovasc. Pharmacol. 31:601-8). Subsequently,subjects were anesthetized, placed on assisted ventilation and athoracotomy performed. The heart and its intrinsic circulation isidentified and a removable suture placed around the most proximalportion of the left anterior descending coronary artery and thenligated. An additional dose of EPO (5000 U/kg) was then given and theocclusion maintained for 30 minutes. At this time, the ligature wasloosened and the animal is maintained under deep anesthesia for anadditional 6 hours and subsequently sacrificed. Immediately after death,the heart was removed and a portion of the affected region (AAR) as wellas unaffected region (septum) was removed and prepared for biochemicalanalyses. Two parameters were assessed, creatine kinase (CK) as ameasure of the survival of myocardium (the lower the CK, the less viablethe tissue) and myeloperoxidase, which is a product of mononuclear cellinfiltrate. The results are shown in FIG. 6A and FIG. 6B. As indicatedin these figures, treatment with EPO results in maintained CK activity,consistent with an increase in tissue viability, and decreased MPOactivity, relative to the control, in both the infarct area (AAR) andthe perfused left ventricle (LV) free wall, indicating that there issignificantly less infiltration by inflammatory cells.

12. EXAMPLE 7 Peripherally Administered EPO Attenuates ExperimentalAllergic Encephalitis

Experimental allergic (or autoimmune) encephalomyelitis (EAE) in rats,is an art accepted animal model for multiple sclerosis (MS). Variousanimal models with EAE have been developed applying immunologic,virologic, toxic and traumatic parameters in order to understandfeatures of MS.

To test whether EPO protects against symptoms of EAE, the followingexperiment was performed. Female Lewis rats, 6-8 weeks of age (CharlesRiver, Calco, Italy) were immunized under light ether anesthesia byinjecting into both hind footpads 50 μg of guinea pig myelin basicprotein (MBP; Sigma, St. Louis, Mo.) in water, emulsified in equalvolumes of complete Freund's adjuvant (CFA, Sigma) with 7 mg/ml ofheat-killed Mycobacterium tuberculosis added to H37Ra (Difco, Detroit,Mich.) in a final volume of 100 μl.

After treatments, rats were assessed daily for signs of experimentalautoimmune encephalomyelitis (EAE) and scored as follows: 0, no disease;1, flaccid tail; 2, ataxia; 3, complete hind limb paralysis with urinaryincontinence. Body weights were also monitored. Rats were administeredEPO (5000 U/kg, IP, once daily) starting on day 3 post-immunization andcontinued until day 18. Control rats received vehicles alone. As shownin FIG. 7, rats treated with EPO demonstrated an improvement in score(i.e., a lower number) and in the duration of the disease. In addition,a marked delay in the onset of symptoms was noticed in rats treated withEPO.

13. EXAMPLE 8 Minimum Effective Dose and Pharmacokinetics of EPORequired for Protection of Excitable Tissue

Optimum and effective dosages of EPO was assessed using the animal modelof focal ischemia stroke described above. As shown in FIG. 8A, an EPOdosage of less than 450 Units/kg body weight was not reliably effectivein protecting excitable tissue from necrotic injury. As shown in FIG.8B, in animal studies, a dose of approximately 5000 Units/kg-body weightdelivered IP to four female mouse subjects resulted in a circulatinglevel of EPO greater than 20,000 mUnits/ml of serum within 5 hours afterits administration, greater than 10,000 mUnits after 10 hours postadministration, but less than 5 Units/ml 24 hours after administration.

14. EXAMPLE 9 CNS Delivery Mediated by Erythropoietin

The experiments presented hereinbelow indicate the successful transportof a molecule conjugated to EPO across the blood-brain barrier and itslocalization inside basement membrane. As shown in FIG. 9A, brainsections were stained with antibodies for EPO receptor (EPO-R), whichshows that brain capillaries express high levels of EPO-R. In order tostudy whether EPO is can be transported across the blood-brain barrier,EPO was labeled with biotin as follows. The volume containing rhEPO wasconcentrated using a Centricon-10 filter (Millipore), and recoverymeasured by reading the absorbance reading at a wavelength of 280 nm.Next, 0.2 mg of long arm biotin (Vector Labs) was dissolved in 100 μl ofDMSO, added to the concentrated rhEPO solution and vortexed immediately.This mixture was then incubated at room temperature for four hours,while gently stirring and protected from light. Unbound biotin wasremoved from the solution by using a Centricon-10 column. BiotinylatedEPO was then administered to animals IP, and 5 hours later the animalswere sacrificed. Brain sections were labeled with avidin coupled toperoxidase, and diaminobenzidine added until sufficient reaction productdeveloped for observation by light microscopy. EPO was found along thesame capillaries that stained positive for EPO-R (FIG. 9B). At latertime points, the biotin label appeared localized within specific neurons(e.g., 17 hours, FIG. 9C). In contrast, if cold EPO was added in 1000time excess to labeled EPO, all specific staining was eliminated. Theresults demonstrate the successful delivery of a systemicallyadministered conjugated EPO compound across the blood brain barrier.

Successful delivery of a systemically administered EPO-biotin conjugateacross the blood brain barrier into the brain demonstrates that othertherapeutic compounds can be delivered across the blood-brain barrier insimilar fashion, by complexing EPO to the compound of interest. As oneexample, brain-derived neurotrophic factor (BNF) can be covalentlycoupled to EPO by carbodiimide coupling using standard procedures. Afterpurification, the conjugate can administered to animals viaintraperitoneal injection. Positive effects of BNF on the centralnervous system can be measured relative to control animals, to measurethe successful transport of this molecule in association with EPO, incontrast to the lack of a central nervous system activity byunconjugated BNF.

The invention is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the invention, and functionally equivalent methods andcomponents are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference herein intheir entireties for all purposes.

1. A method for enhancing the function of normal or abnormal excitabletissue in a mammal comprising administering peripherally to said mammala peripherally effective excitable tissue enhancing amount of an EPO, anEPO receptor activity modulator, an EPO-activated receptor modulator, orcombination thereof.
 2. The method of claim 1 wherein said enhancing thefunction of excitable tissue results in the enhancement of associativelearning or memory.
 3. The method of claim 1 wherein said enhancing thefunction of excitable tissue is used in the treatment of mood disorders,anxiety disorders, depression, autism, attention deficit hyperactivitydisorder, Alzheimer's disease, aging or cognitive dysfunction.
 4. Themethod of claim 1 wherein said excitable tissue is central nervoussystem tissue or peripheral nervous system tissue.
 5. The method ofclaim 1 wherein said administration comprises oral, topical,intraluminal or by inhalation or parenteral administration.
 6. Themethod of claim 5 wherein said parenteral administration is intravenous,intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosalor intradermal.
 7. The method of claim 1 wherein said administration isacute or chronic.
 8. The method of claim 1 wherein said EPO isnonerythropoietic.
 9. The method of claim 1 wherein said EPO isadministered at a dose greater than the dose necessary to maximallystimulate erythropoiesis.
 10. The method of claim 1, wherein said EPO iserythropoietin, an erythropoietin analog, an erythropoietin mimetic, anerythropoietin fragment, a hybrid erythropoietin molecule, anerythropoietin receptor-binding molecule, an erythropoietin agonist, arenal erythropoietin, a brain erythropoietin, an oligomer thereof, amultimer thereof, a mutein thereof, a congener thereof, anaturally-occurring form thereof, a synthetic form thereof, arecombinant form thereof, or a combination thereof.
 11. The method ofclaim 10 wherein said EPO receptor-binding molecule is an antibody tothe erythropoietin receptor.